Crystalline form of a 4-[2-(2-fluorophenoxymethyl)phenyl]piperidine compound

ABSTRACT

The invention provides a crystalline hydrochloride salt of 4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine. This invention also provides pharmaceutical compositions comprising the crystalline salt, processes and intermediates for preparing the crystalline salt, and methods of using the crystalline salt to treat diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/728,079, filed on Jun. 2, 2015; which application is a divisional ofU.S. Ser. No. 14/060,846, filed Oct. 23, 2013 (now U.S. Pat. No.9,073,859 B2); which application is a divisional of U.S. Ser. No.13/632,588, filed Oct. 1, 2012 (now U.S. Pat. No. 8,592,596 B2); whichapplication is a divisional of U.S. Ser. No. 12/617,838, filed Nov. 13,2009 (now U.S. Pat. No. 8,304,433 B2); which application claims thebenefit of U.S. Provisional Application No. 61/114,541, filed on Nov.14, 2008; the entire disclosures of which are incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a novel crystalline form of a4-[2-(2-fluorophenoxymethyl)phenyl]piperidine compound, which hasactivity as a serotonin (5-HT) and norepinephrine (NE) reuptakeinhibitor. This invention also relates to pharmaceutical compositionscomprising the crystalline compound or prepared from such compound,processes and intermediates for preparing the crystalline compound, andmethods of using such compound to treat a pain disorder, such asneuropathic pain, and other ailments.

State of the Art

Pain is an unpleasant sensory and emotional experience associated withactual or potential tissue damage, or described in terms of such damage(International Association for the Study of Pain (IASP), PainTerminology). Chronic pain persists beyond acute pain or beyond theexpected time for an injury to heal (American Pain Society. “PainControl in the Primary Care Setting.” 2006:15). Neuropathic pain is paininitiated or caused by a primary lesion or dysfunction in the nervoussystem. Peripheral neuropathic pain occurs when the lesion ordysfunction affects the peripheral nervous system and centralneuropathic pain when the lesion or dysfunction affects the centralnervous system (IASP).

Several types of therapeutic agents are currently used to treatneuropathic pain including, for example, tricyclic antidepressants,serotonin and norepinephrine reuptake inhibitors, calcium channelligands (e.g., gabapentin and pregabalin), topical lidocaine, and opioidagonists (e.g., morphine, oxycodone, methadone, levorphanol andtramadol).

4-[2-(2,4,6-Trifluorophenoxymethyl)phenyl]piperidine, described herein,inhibits the reuptake of both serotonin and norepinephrine by binding tothe serotonin and norepinephrine transporters. When preparing compoundsfor long term storage and when preparing pharmaceutical compositions andformulations, it is often desirable to have a crystalline form of thetherapeutic agent that is neither hygroscopic nor deliquescent. It isalso advantageous to have a crystalline form that has a relatively highmelting point (i.e. greater than about 150° C.), which allows thematerial to be processed, for example, micronized, without significantdecomposition. Accordingly, a need exists for a stable, non-deliquescentform of 4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine which hasan acceptable level of hygroscopicity and a relatively high meltingpoint.

SUMMARY OF THE INVENTION

The present invention relates to a crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine.

One aspect of the invention relates to processes for preparing acrystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine. In one embodiment,a process for preparing a crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine comprises the stepsof: a) treating a hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine with ethyl acetateand ethanol to complete dissolution; b) cooling to effectcrystallization; c) isolating the resulting solids to yield thecrystalline hydrochloride salt of the invention. In another embodiment,this crystalline hydrochloride salt is further d) treated withisopropanol and water to complete dissolution; e) cooled to effectcrystallization; and f) the resulting solids isolated to yield thecrystalline hydrochloride salt of the invention.

Another aspect of the invention relates to a process for purifying4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine. In one embodiment,this process comprises forming a crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl] piperidine. The inventionalso relates to products prepared by the processes described herein.

One aspect of the invention relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a crystallinehydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine. Such compositionsmay optionally contain other active agents such as anti-Alzheimer'sagents, anticonvulsants, antidepressants, anti-Parkinson's agents, dualserotonin-norepinephrine reuptake inhibitors, non-steroidalanti-inflammatory agents, norepinephrine reuptake inhibitors, opioidagonists, opioid antagonists, selective serotonin reuptake inhibitors,sodium channel blockers, sympatholytics, and combinations thereof.Accordingly, in yet another aspect of the invention, a pharmaceuticalcomposition comprises the crystalline salt of the invention, a secondactive agent, and a pharmaceutically acceptable carrier. Another aspectof the invention relates to a combination of active agents, comprisingthe crystalline salt of the invention and a second active agent. Thecrystalline salt of the invention can be formulated together orseparately from the additional agent(s). When formulated separately, apharmaceutically acceptable carrier may be included with the additionalagent(s). Thus, yet another aspect of the invention relates to acombination of pharmaceutical compositions, the combination comprising:a first pharmaceutical composition comprising the crystalline salt ofthe invention and a first pharmaceutically acceptable carrier; and asecond pharmaceutical composition comprising a second active agent and asecond pharmaceutically acceptable carrier. The invention also relatesto a kit containing such pharmaceutical compositions, for example wherethe first and second pharmaceutical compositions are separatepharmaceutical compositions.

4-[2-(2,4,6-Trifluorophenoxymethyl)phenyl]piperidine possesses serotoninreuptake inhibitory activity and norepinephrine reuptake inhibitoryactivity. The crystalline hydrochloride salt of this compound isexpected to have the same activity and thus the same utility as atherapeutic agent for treating patients suffering from a disease ordisorder that is treated by the inhibition of the serotonin and/or thenorepinephrine transporter. Thus, one aspect of the invention relates toa method of treating: a pain disorder such as neuropathic pain orfibromyalgia; a depressive disorder such as major depression; anaffective disorder such as an anxiety disorder; attention deficithyperactivity disorder; a cognitive disorder such as dementia; stressurinary incontinence; chronic fatigue syndrome; obesity; or vasomotorsymptoms associated with menopause, comprising administering to apatient a therapeutically effective amount of the crystalline compoundof the invention.

Yet another aspect of the invention relates to the use of thecrystalline compound of the invention for the manufacture ofmedicaments, especially for the manufacture of medicaments useful fortreating pain disorders, depressive disorders, affective disorders,attention deficit hyperactivity disorder, cognitive disorders, stressurinary incontinence, for inhibiting serotonin reuptake in a mammal, orfor inhibiting norepinephrine reuptake in a mammal. Other aspects andembodiments of the invention are disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a powder x-ray diffraction (PXRD) pattern of thecrystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine.

FIG. 2 shows a differential scanning calorimetry (DSC) thermograph and athermal gravimetric analysis (TGA) trace.

FIG. 3 shows a dynamic moisture sorption (DMS) profile.

FIG. 4 is a micrographic image.

FIG. 5 is a microscopic image of a unit cell crystal.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine. Surprisingly, thiscrystalline compound has been found not to be deliquescent, even whenexposed to atmospheric moisture. Additionally, this crystalline compoundhas an acceptable level of hygroscopicity and a high melting point.

The single crystal of the drug molecule exhibits a monoclinic symmetry(P2₁C space group), with the following unit-cell parameters: A=C=90°,B=104.595°; a=11.631 Å, b=7.057 Å, c=42.532 Å. While not wishing to belimited by theory, based upon the X-ray crystallographic data, thecrystal is believed to be built around the water molecule and the watercontent was determined from the water occupancy factor in the unit cellusing the thermal motion parameters of the atoms and overall observedstructure factors.

Thus, in one embodiment, the crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine may contain acertain amount of water that is absorbed on the surface of thecrystalline material or forms part of the crystalline structure.

In one particular embodiment, the crystalline hydrochloride salt maycontain an amount of water ranging from about 0.2 wt % to about 0.8 wt %that is absorbed on the crystal surface; and in another embodiment fromabout 0.4 wt % to about 0.6 wt %.

In yet another particular embodiment, the crystalline hydrochloride saltmay contain from about 0.25 to about 0.50 moles of water that form partof the crystalline structure; and in another embodiment from about 0.30to about 0.40 moles. In one exemplary embodiment, there are about 0.32moles of water present for every one mole of the4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine hydrochloride salt.

Definitions

When describing the compounds, compositions, methods and processes ofthe invention, the following terms have the following meanings unlessotherwise indicated. Additionally, as used herein, the singular forms“a,” “an” and “the” include the corresponding plural forms unless thecontext of use clearly dictates otherwise. The terms “comprising”,“including,” and “having” are intended to be inclusive and mean thatthere may be additional elements other than the listed elements. Allnumbers expressing quantities of ingredients, properties such asmolecular weight, reaction conditions, and so forth used herein are tobe understood as being modified in all instances by the term “about,”unless otherwise indicated. Accordingly, the numbers set forth hereinare approximations that may vary depending upon the desired propertiessought to be obtained by the present invention. At least, and not as anattempt to limit the application of the doctrine of equivalents to thescope of the claims, each number should at least be construed in lightof the reported significant digits and by applying ordinary roundingtechniques.

As used herein, the phrase “of the formula”, “having the formula” or“having the structure” is not intended to be limiting and is used in thesame way that the term “comprising” is commonly used.

The term “melting point” as used herein means the temperature at whichthe maximum endothermic heat flow is observed by differential scanningcalorimetry, for the thermal transition that corresponds to thesolid-to-liquid phase change.

The term “pharmaceutically acceptable” refers to a material that is notbiologically or otherwise unacceptable when used in the invention. Forexample, the term “pharmaceutically acceptable carrier” refers to amaterial that can be incorporated into a composition and administered toa patient without causing unacceptable biological effects or interactingin an unacceptable manner with other components of the composition. Suchpharmaceutically acceptable materials typically have met the requiredstandards of toxicological and manufacturing testing, and include thosematerials identified as suitable inactive ingredients by the U.S. Foodand Drug Administration.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need thereof,i.e., the amount of drug needed to obtain the desired therapeuticeffect. For example, a therapeutically effective amount for treatingneuropathic pain is an amount of compound needed to, for example,reduce, suppress, eliminate or prevent the symptoms of neuropathic painor to treat the underlying cause of neuropathic pain. On the other hand,the term “effective amount” means an amount sufficient to obtain adesired result, which may not necessary be a therapeutic result. Forexample, when studying a system comprising a norepinephrine transporter,an “effective amount” may be the amount needed to inhibit norepinephrinereuptake.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as neuropathic pain)in a patient, such as a mammal (particularly a human), that includes oneor more of the following: (a) preventing the disease or medicalcondition from occurring, i.e., prophylactic treatment of a patient; (b)ameliorating the disease or medical condition, i.e., eliminating orcausing regression of the disease or medical condition in a patient; (c)suppressing the disease or medical condition, i.e., slowing or arrestingthe development of the disease or medical condition in a patient; or (d)alleviating the symptoms of the disease or medical condition in apatient. For example, the term “treating neuropathic pain” would includepreventing neuropathic pain from occurring, ameliorating neuropathicpain, suppressing neuropathic pain, and alleviating the symptoms ofneuropathic pain. The term “patient” is intended to include thosemammals, such as humans, that are in need of treatment or diseaseprevention, that are presently being treated for disease prevention ortreatment of a specific disease or medical condition, as well as testsubjects in which compounds of the invention are being evaluated orbeing used in a assay, for example an animal model.

All other terms used herein are intended to have their ordinary meaningas understood by those of ordinary skill in the art to which theypertain.

The crystalline compound of the invention can be synthesized fromreadily available starting materials as described below and in theExamples. It will be appreciated that where typical or preferred processconditions (i.e., reaction temperatures, times, mole ratios ofreactants, solvents, pressures, etc.) are given, other processconditions can also be used unless otherwise stated. It will beappreciated that while specific process conditions (i.e. crystallizationtemperatures, times, mole ratios of reactants, solvents, pressures,etc.) are given, other process conditions can also be used unlessotherwise stated. In some instances, reactions or crystallizations wereconducted at room temperature and no actual temperature measurement wastaken. It is understood that room temperature can be taken to mean atemperature within the range commonly associated with the ambienttemperature in a laboratory environment, and will typically be in therange of about 25° C. to about 50° C. In other instances, reactions orcrystallizations were conducted at room temperature and the temperaturewas actually measured and recorded.

Generally, the crystallization is conducted in a suitable inert diluentor solvent system, examples of which include, but are not limited to,methanol, ethanol, isopropanol, isobutanol, ethyl acetate, acetonitrile,dichloromethane, methyl t-butyl ether, and the like, and mixturesthereof, optionally containing water. Upon completion of thecrystallization, the crystalline compound can be isolated from thereaction mixture by any conventional means such as precipitation,concentration, centrifugation and the like.

The 4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine employed in theinvention can be readily prepared from commercially available startingmaterials and reagents using the procedures described in the Examples.The molar ratios described in the methods of the invention can bereadily determined by various methods available to those skilled in theart. For example, such molar ratios can be readily determined by ¹H NMR.Alternatively, elemental analysis and HPLC methods can be used todetermine the molar ratio.

In general, the crystalline compound of the invention can be prepared bytreating 4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine with aninert diluent to complete dissolution. Suitable inert diluents includeby way of illustration and not limitation, acetone, acetonitrile, ethylacetate, methyl ethyl ketone, methanol, ethanol, isopropanol, water, andso forth. Other suitable inert diluents include by way of illustrationand not limitation, combinations of inert diluents such as acetone withwater, acetonitrile with water, ethanol and ethyl acetate, methanol andwater, and isopropanol and water. In one particular embodiment, theinert diluent is ethyl acetate or a combination of isopropanol withwater. Generally, dissolution is conducted at a temperature ranging fromabout 50° C. to about 90° C., in one embodiment at a temperature rangingfrom about 60-80° C., and in another embodiment at a temperature rangingfrom about 65-75° C. The solution is then cooled to form the crystallinecompound of the invention. In one particular embodiment, the solution iscooled to about 20-30° C. such as 25° C. After a suitable amount oftime, crystals will be observed. In one embodiment, crystals areobserved after a period of about 1 hour. After crystals are observed,the volume of the mother liquor can be reduced and the crystals isolatedand dried. In one embodiment, once crystals are observed, crystals areallowed to develop for a period of about 12-24 hours prior to isolation.

In one embodiment, the crystalline compound of the invention is preparedby treating the hydrochloride salt4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine with ethyl acetateand ethanol to complete dissolution and cooling to effectcrystallization. The crystals are then isolated to yield a crystallinehydrochloride salt having purity typically greater than 99%. Typically,this process can be conducted at any of the aforementioned temperatureranges. For example, dissolution can be done at about 65-70° C.,followed by cooling to room temperature.

The same crystalline form can be prepared with a slightly higher purityand better hydroscopic properties by recrystallization. Thus, in anotherembodiment, the crystalline hydrochloride salt prepared using ethylacetate and ethanol is recrystallized with isopropanol and water, i.e.,treated with isopropanol and water to complete dissolution and cooled toeffect crystallization. The resulting solids can then be isolated anddried to yield the crystalline compound of the invention. Typically,this process can be conducted at any of the aforementioned temperatureranges. For example, dissolution can be done at about 75° C., followedby cooling to room temperature. The volume ratio of isopropanol to waterwill typically be in the range of about 9:1 to about 21:1, for example,in one embodiment about 10:1 and in another embodiment about 20:1.

The hydrochloride salt starting material can be prepared by techniquesthat are well known in the art, and examples are provided in theExamples herein. For example,4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine-1-carboxylic acidt-butyl ester can be deprotected using hydrochloric acid in ethanol toyield the hydrochloride salt.

CRYSTALLINE PROPERTIES

Among other advantages, it has been discovered that forming acrystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine, is useful forpurifying the compound itself. For example, the crystallinehydrochloride salt of the invention has a purity of greater than 99.0%.

As is well known in the field of powder x-ray diffraction, relative peakheights of PXRD spectra are dependent on a number of factors relating tosample preparation and instrument geometry, while peak positions arerelatively insensitive to experimental details. A PXRD pattern wasobtained as set forth in Example 4. Thus, in one embodiment, thecrystalline compound of the invention is characterized by a PXRD patternhaving certain peak positions.

The crystalline compound is characterized by a PXRD pattern in which thepeak positions are substantially in accordance with those shown inFIG. 1. Those peaks are listed below, in order of descending relativeintensity. All PXRD peak intensities were corrected by subtracting thecorresponding background intensity for each peak.

I % 2-Theta 100 17.16 66 16.06 55 10.22 51 26.32 43 18.38 33 21.78 3213.18 30 27.24 26 29.60 16 4.44 24 31.94 19 23.76 22 8.11Thus, in one embodiment, the crystalline compound is characterized by apowder x-ray diffraction (PXRD) pattern comprising diffraction peaks at20 values of 4.44±0.20, 10.22±0.20, 17.16±0.20, and 21.78±0.20; andfurther characterized by having one or more additional diffraction peaksat 20 values selected from 8.11±0.20, 13.18±0.20, 16.06±0.20,18.38±0.20, 23.76±0.20, 26.32±0.20, 27.24±0.20, 29.60±0.20, and31.94±0.20.

A differential scanning calorimetry (DSC) trace was obtained as setforth in Example 5. Thus, in one embodiment, the crystalline compound ischaracterized by its DSC thermograph. In one embodiment, the crystallinecompound is characterized by a DSC thermograph which shows a meltingpoint of about 196.9° C., with no significant thermal decompositionbelow about 200.0° C., as seen in FIG. 2.

Thermogravimetric analysis (TGA) was performed on the crystallinecompound as described in Example 5. Thus, in one embodiment, thecrystalline compound is characterized by its TGA trace. In oneembodiment, the crystalline compound is characterized by a TGA tracewhich shows a loss of solvents and/or water (about 0.5%) at temperaturesbelow about 150.0° C., as seen in FIG. 2.

The crystalline compound of the invention has been demonstrated to havea reversible sorption/desorption profile with acceptable levels ofhygroscopicity. For example, the crystalline compound has no or minimalhygroscopicity propensity, and has exhibited less than about 2.0% weightgain when exposed to up to 90% relative humidity, as seen in FIG. 3.

These properties of the crystalline compound of the invention arefurther illustrated in the Examples below.

UTILITY

4-[2-(2,4,6-Trifluorophenoxymethyl)phenyl]piperidine possesses serotoninand norepinephrine reuptake inhibitory activity. Thus, this compound, aswell as of the crystalline compound of the invention, is expected tohave therapeutic utility as a combined serotonin and norepinephrinereuptake inhibitor (SNRI).

The inhibition constant (K_(i)) of a compound is the concentration ofcompeting ligand in a competition assay that would occupy 50% of thetransporters if no radioligand were present. K_(i) values can bedetermined from radioligand competition binding studies with³H-nisoxetine (for the norepinephrine transporter, NET) and³H-citalopram (for the serotonin transporter, SERT), as described inAssay 1. These K_(i) values are derived from IC₅₀ values in the bindingassay using the Cheng-Prusoff equation and the K_(d) of the radioligand(Cheng & Prusoff (1973) Biochem. Pharmacol. 22(23):3099-3108).Functional IC₅₀ values can be determined in the functional inhibition ofuptake assays described in Assay 2. These IC₅₀ values can be convertedto K_(i) values using the Cheng-Prusoff equation and the K_(m) of thetransmitter for the transporter. It is noted however, that the uptakeassay conditions described in Assay 2 are such that the IC₅₀ values arevery close to the K_(i) values, should a mathematical conversion bedesired, since the neurotransmitter concentration (5-HT or NE) used inthe assay is well below its K_(m) for the respective transporter.

Exemplary assays to determine the serotonin and/or norepinephrinereuptake inhibiting activity of the crystalline compound of theinvention include by way of illustration and not limitation, assays thatmeasure SERT and NET binding, for example, as described in Assay 1. Inaddition, it is useful to understand the level of DAT binding and uptakein an assay such as that described in Assay 1. Useful secondary assaysinclude neurotransmitter uptake assays to measure competitive inhibitionof serotonin and norepinephrine uptake into cells expressing therespective human or rat recombinant transporter (hSERT, hNET, or hDAT)as described in Assay 2, and ex vivo radioligand binding andneurotransmitter uptake assays that are used to determine the in vivooccupancy of SERT, NET and DAT in tissue as described in Assay 3. Otherassays that are useful to evaluate pharmacological properties includethose listed in Assay 4. Exemplary in vivo assays include the formalinpaw test described in Assay 5, which is a reliable predictor of clinicalefficacy for the treatment of neuropathic pain, and the spinal nerveligation model described in Assay 6. The aforementioned assays areuseful in determining the therapeutic utility, for example, theneuropathic pain relieving activity, of the crystalline compound of theinvention. Other properties and utilities of the crystalline compound ofthe invention can be demonstrated using various in vitro and in vivoassays well-known to those skilled in the art.

The crystalline compound of the invention is expected to be useful forthe treatment and/or prevention of medical conditions in which theregulation of monoamine transporter function is implicated, inparticular those conditions mediated by or responsive to the inhibitionof serotonin and norepinephrine reuptake. Thus it is expected thatpatients suffering from a disease or disorder that is treated by theinhibition of the serotonin and/or the norepinephrine transporter can betreated by administering a therapeutically effective amount of thecrystalline compound of the invention. Such medical conditions include,by way of example, pain disorders such as neuropathic pain,fibromyalgia, and chronic pain, depressive disorders such as majordepression, affective disorders such as an anxiety disorder, attentiondeficit hyperactivity disorder, cognitive disorders such as dementia,and stress urinary incontinence.

The amount of active agent administered per dose or the total amountadministered per day may be predetermined or it may be determined on anindividual patient basis by taking into consideration numerous factors,including the nature and severity of the patient's condition, thecondition being treated, the age, weight, and general health of thepatient, the tolerance of the patient to the active agent, the route ofadministration, pharmacological considerations such as the activity,efficacy, pharmacokinetics and toxicology profiles of the active agentand any secondary agents being administered, and the like. Treatment ofa patient suffering from a disease or medical condition (such asneuropathic pain) can begin with a predetermined dosage or a dosagedetermined by the treating physician, and will continue for a period oftime necessary to prevent, ameliorate, suppress, or alleviate thesymptoms of the disease or medical condition. Patients undergoing suchtreatment will typically be monitored on a routine basis to determinethe effectiveness of therapy. For example, in treating neuropathic pain,a measure of the effectiveness of treatment may involve assessment ofthe patient's quality of life, e.g., improvements in the patient'ssleeping patterns, work attendance, ability to exercise and beambulatory, etc. Pain scales, operating on a point basis, may also beused to help evaluate a patient's pain level. Indicators for the otherdiseases and conditions described herein, are well-known to thoseskilled in the art, and are readily available to the treating physician.Continuous monitoring by the physician will insure that the optimalamount of active agent will be administered at any given time, as wellas facilitating the determination of the duration of treatment. This isof particular value when secondary agents are also being administered,as their selection, dosage, and duration of therapy may also requireadjustment. In this way, the treatment regimen and dosing schedule canbe adjusted over the course of therapy so that the lowest amount ofactive agent that exhibits the desired effectiveness is administeredand, further, that administration is continued only so long as isnecessary to successfully treat the disease or medical condition.

Pain Disorders

SNRIs have been shown to have a beneficial effect on pain such aspainful diabetic neuropathy (duloxetine, Goldstein et al. (2005) Pain116:109-118; venlafaxine, Rowbotham et al. (2004) Pain 110:697-706),fibromyalgia (duloxetine, Russell et al. (2008) Pain 136(3):432-444;milnacipran, Vitton et al. (2004) Human Psychopharmacology 19:S27-S35),and migraine (venlafaxine, Ozyalcin et al. (2005) Headache45(2):144-152). Thus, one embodiment of the invention relates to amethod for treating a pain disorder, comprising administering to apatient a therapeutically effective amount of the crystalline compoundof the invention. Typically, the therapeutically effective amount willbe the amount that is sufficient to relieve the pain. Exemplary paindisorders include, by way of illustration, acute pain, persistent pain,chronic pain, inflammatory pain, and neuropathic pain. Morespecifically, these include pain associated with or caused by:arthritis; back pain including chronic low back pain; cancer, includingtumor related pain (e.g., bone pain, headache, facial pain or visceralpain) and pain associated with cancer therapy (e.g., post-chemotherapysyndrome, chronic post-surgical pain syndrome and post-radiationsyndrome); carpal tunnel syndrome; fibromyalgia; headaches includingchronic tension headaches; inflammation associated with polymyalgia,rheumatoid arthritis and osteoarthritis; migraine; neuropathic painincluding complex regional pain syndrome; overall pain; post-operativepain; shoulder pain; central pain syndromes, including post-stroke pain,and pain associated with spinal cord injuries and multiple sclerosis;phantom limb pain; pain associated with Parkinson's disease; andvisceral pain (e.g., irritable bowel syndrome). Of particular interestis the treatment of neuropathic pain, which includes diabetic peripheralneuropathy (DPN), HIV-related neuropathy, post-herpetic neuralgia (PHN),and chemotherapy-induced peripheral neuropathy. When used to treat paindisorders such as neuropathic pain, compounds of the invention may beadministered in combination with other therapeutic agents, includinganticonvulsants, antidepressants, muscle relaxants, NSAIDs, opioidagonists, opioid antagonists, selective serotonin reuptake inhibitors,sodium channel blockers, and sympatholytics. Exemplary compounds withinthese classes are described herein.

Depressive Disorders

Another embodiment of the invention relates to a method of treating adepressive disorder, comprising administering to a patient atherapeutically effective amount of the crystalline compound of theinvention. Typically, the therapeutically effective amount will be theamount that is sufficient to alleviate depression and provide a sense ofgeneral well-being. Exemplary depressive disorders include, by way ofillustration and not limitation: depression associated with Alzheimer'sdisease, bipolar disorder, cancer, child abuse, infertility, Parkinson'sdisease, postmyocardial infarction, and psychosis; dysthymia; grumpy orirritable old man syndrome; induced depression; major depression;pediatric depression; postmenopausal depression; post partum depression;recurrent depression; single episode depression; and subsyndromalsymptomatic depression. Of particular interest is the treatment of majordepression. When used to treat depressive disorders, compounds of theinvention may be administered in combination with other therapeuticagents, including antidepressants and dual serotonin-norepinephrinereuptake inhibitors. Exemplary compounds within these classes aredescribed herein.

Affective Disorders

Another embodiment of the invention relates to a method of treating anaffective disorder, comprising administering to a patient atherapeutically effective amount of the crystalline compound of theinvention. Exemplary affective disorders include, by way of illustrationand not limitation: anxiety disorders such as general anxiety disorder;avoidant personality disorder; eating disorders such as anorexianervosa, bulimia nervosa and obesity; obsessive compulsive disorder;panic disorder; personality disorders such as avoidant personalitydisorder and attention deficit hyperactivity disorder (ADHD);post-traumatic stress syndrome; phobias such as agoraphobia, as well assimple and other specific phobias, and social phobia; premenstrualsyndrome; psychotic disorders, such as schizophrenia and mania; seasonalaffective disorder; sexual dysfunction, including premature ejaculation,male impotence, and female sexual dysfunction such as female sexualarousal disorder; social anxiety disorder; and substance abusedisorders, including chemical dependencies such as addictions toalcohol, benzodiazepines, cocaine, heroin, nicotine and phenobarbital,as well as withdrawal syndromes that may arise from these dependencies.When used to treat affective disorders, compounds of the invention maybe administered in combination with other therapeutic agents, includingantidepressants. Exemplary compounds within these classes are describedherein.

Atomoxetine, which is 10-fold NET selective, is approved for attentiondeficit hyperactivity disorder (ADHD) therapy, and clinical studies haveshown that the SNRI, venlafaxine, can also have a beneficial effect intreating ADHD (Mukaddes et al. (2002) Eur. Neuropsychopharin. 12(Supp3):421). Thus, the crystalline compound of the invention is alsoexpected to be useful in methods for treating attention deficithyperactivity disorder by administering to a patient a therapeuticallyeffective amount of the crystalline compound of the invention. When usedto treat depression, the crystalline compound of the invention may beadministered in combination with other therapeutic agents, includingantidepressants. Exemplary compounds within these classes are describedherein.

Cognitive Disorders

Another embodiment of the invention relates to a method of treating acognitive disorder, comprising administering to a patient atherapeutically effective amount of the crystalline compound of theinvention. Exemplary cognitive disorders include, by way of illustrationand not limitation: dementia, which includes degenerative dementia(e.g., Alzheimer's disease, Creutzfeldt-Jakob disease, Huntingdon'schorea, Parkinson's disease, Pick's disease, and senile dementia),vascular dementia (e.g., multi-infarct dementia), and dementiaassociated with intracranial space occupying lesions, trauma, infectionsand related conditions (including HIV infection), metabolism, toxins,anoxia and vitamin deficiency; and mild cognitive impairment associatedwith ageing, such as age associated memory impairment, amnesiac disorderand age-related cognitive decline. When used to treat cognitivedisorders, the crystalline compound of the invention may be administeredin combination with other therapeutic agents, including anti-Alzheimer'sagents and anti-Parkinson's agents. Exemplary compounds within theseclasses are described herein.

Other Disorders

SNRIs have also been shown to be effective for the treatment of stressurinary incontinence (Dmochowski (2003) Journal of Urology 170(4):1259-1263). Thus, another embodiment of the invention relates to amethod for treating stress urinary incontinence, comprisingadministering to a patient a therapeutically effective amount of thecrystalline compound of the invention. When used to treat stress urinaryincontinence, compounds of the invention may be administered incombination with other therapeutic agents, including anticonvulsants.Exemplary compounds within these classes are described herein.

Duloxetine, an SNRI, is undergoing clinical trials for evaluating itsefficacy in treating chronic fatigue syndrome, and has recently beenshown to be effective in treating fibromyalgia (Russell et al. (2008)Pain 136(3):432-444). The crystalline compound of the invention, due toits expected ability to inhibit SERT and NET, is also expected to havethis utility, and another embodiment of the invention relates to amethod for treating chronic fatigue syndrome, comprising administeringto a patient a therapeutically effective amount of the crystallinecompound of the invention.

Sibutramine, a norepinephrine and dopamine reuptake inhibitor, has beenshown to be useful in treating obesity (Wirth et al. (2001) JAMA286(11):1331-1339). The crystalline compound of the invention, due toits expected ability to inhibit NET, is also expected to have thisutility, and another embodiment of the invention relates to a method fortreating obesity, comprising administering to a patient atherapeutically effective amount of the crystalline compound of theinvention.

Desvenlafaxine, an SNRI, has been shown to relieve vasomotor symptomsassociated with menopause (Deecher et al. (2007) Endocrinology148(3):1376-1383). The crystalline compound of the invention, due to itsexpected ability to inhibit SERT and NET, is also expected to have thisutility, and another embodiment of the invention relates to a method fortreating vasomotor symptoms associated with menopause, comprisingadministering to a patient a therapeutically effective amount of thecrystalline compound of the invention.

Research Tools

Since the crystalline compound of the invention is expected to possessboth serotonin reuptake inhibition activity and norepinephrine reuptakeinhibition activity, this compound is also expected to find utility as aresearch tool for investigating or studying biological systems orsamples having serotonin or norepinephrine transporters. Any suitablebiological system or sample having serotonin and/or norepinephrinetransporters may be employed in such studies which may be conductedeither in vitro or in vivo. Representative biological systems or samplessuitable for such studies include, but are not limited to, cells,cellular extracts, plasma membranes, tissue samples, isolated organs,mammals (such as mice, rats, guinea pigs, rabbits, dogs, pigs, humans,and so forth), and the like, with mammals being of particular interest.In one particular embodiment of the invention, serotonin reuptake in amammal is inhibited by administering a serotonin reuptake-inhibitingamount of the crystalline compound of the invention. In anotherparticular embodiment, norepinephrine reuptake in a mammal is inhibitedby administering a norepinephrine reuptake-inhibiting amount of thecrystalline compound of the invention. The crystalline compound of theinvention can also be used as a research tool by conducting biologicalassays using such compound.

When used as a research tool, a biological system or sample comprising aserotonin transporter and/or a norepinephrine transporter is typicallycontacted with a serotonin reuptake-inhibiting or norepinephrinereuptake-inhibiting amount of the crystalline compound of the invention.After the biological system or sample is exposed to the compound, theeffects of inhibiting serotonin reuptake and/or norepinephrine reuptakeare determined using conventional procedures and equipment. Exposureencompasses contacting cells or tissue with the compound, administeringthe compound to a mammal, for example by i.p. or i.v. administration,and so forth. This determining step may comprise measuring a response,i.e., a quantitative analysis or may comprise an observation, i.e., aqualitative analysis. Measuring a response involves, for example,determining the effects of the compound on the biological system orsample using conventional procedures and equipment, such as serotoninand norepinephrine reuptake assays. The assay results can be used todetermine the activity level as well as the amount of compound necessaryto achieve the desired result, i.e., a serotonin reuptake-inhibiting anda norepinephrine reuptake-inhibiting amount.

Additionally, the crystalline compound of the invention can be used as aresearch tool for evaluating other chemical compounds, and thus is alsouseful in screening assays to discover, for example, new compoundshaving both serotonin reuptake-inhibiting activity and norepinephrinereuptake-inhibiting activity. In this manner, the crystalline compoundof the invention is used as a standard in an assay to allow comparisonof the results obtained with a test compound and with the crystallinecompound of the invention to identify those test compounds that haveabout equal or superior reuptake-inhibiting activity, if any. Forexample, reuptake data for a test compound or a group of test compoundsis compared to the reuptake data for the crystalline compound of theinvention to identify those test compounds that have the desiredproperties, e.g., test compounds having reuptake-inhibiting activityabout equal or superior to the crystalline compound of the invention, ifany. This aspect of the invention includes, as separate embodiments,both the generation of comparison data (using the appropriate assays)and the analysis of the test data to identify test compounds ofinterest. Thus, a test compound can be evaluated in a biological assay,by a method comprising the steps of: (a) conducting a biological assaywith a test compound to provide a first assay value; (b) conducting thebiological assay with the crystalline compound of the invention toprovide a second assay value; wherein step (a) is conducted eitherbefore, after or concurrently with step (b); and (c) comparing the firstassay value from step (a) with the second assay value from step (b).Exemplary biological assays include serotonin and norepinephrinereuptake assays.

Pharmaceutical Compositions and Formulations

The crystalline compound of the invention is typically administered to apatient in the form of a pharmaceutical composition or formulation. Suchpharmaceutical compositions may be administered to the patient by anyacceptable route of administration including, but not limited to, oral,rectal, vaginal, nasal, inhaled, topical (including transdermal) andparenteral modes of administration. However, it will be understood bythose skilled in the art that, once the crystalline compound of theinvention has been formulated, it may no longer be in crystalline form,i.e., it may be dissolved in a suitable carrier. Further, thecrystalline compound of the invention may be administered, for exampleorally, in multiple doses per day (e.g., twice, three times or fourtimes daily), in a single daily dose, in a twice daily dose, in a singleweekly dose, and so forth.

Accordingly, in one embodiment, the invention relates to apharmaceutical composition comprising a pharmaceutically acceptablecarrier and the crystalline compound of the invention. The compositionsmay contain other therapeutic and/or formulating agents if desired. Whendiscussing compositions, the “crystalline compound of the invention” mayalso be referred to herein as the “active agent,” to distinguish it fromother components of the formulation, such as the carrier.

Pharmaceutical compositions of the invention typically contain atherapeutically effective amount of the crystalline compound of theinvention. Those skilled in the art will recognize, however, that apharmaceutical composition may contain more than a therapeuticallyeffective amount, i.e., bulk compositions, or less than atherapeutically effective amount, i.e., individual unit doses designedfor multiple administration to achieve a therapeutically effectiveamount. Typically, the composition will contain from about 0.01-95 wt %of active agent, including, from about 0.01-30 wt %, such as from about0.01-10 wt %, with the actual amount depending upon the formulationitself, the route of administration, the frequency of dosing, and soforth. In one embodiment, a composition suitable for an oral dosageform, for example, may contain about 5-70 wt %, or from about 10-60 wt %of active agent. In one exemplary embodiment, a pharmaceuticalcomposition contains from about 1 to 20 mg of active agent, includingfrom about 1 to 15 mg of active agent and from about 1 to 10 mg ofactive agent. In another exemplary embodiment, a pharmaceuticalcomposition contains from about 5 to 20 mg of active agent, includingfrom about 7.5 to 15 mg of active agent. For example the active agentmay be formulated in 1 mg and 10 mg unit doses.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of the invention. The choice of a particular carrier orexcipient, or combinations of carriers or excipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable composition for a particular mode of administration iswell within the scope of those skilled in the pharmaceutical arts.Additionally, carriers or excipients used in such compositions arecommercially available. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following:sugars, such as lactose, glucose and sucrose; starches, such as cornstarch and potato starch; cellulose, such as microcrystalline cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose and cellulose acetate; powdered tragacanth; malt; gelatin;talc; excipients, such as cocoa butter and suppository waxes; oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters,such as ethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; compressed propellant gases, such aschlorofluorocarbons and hydrofluorocarbons; and other non-toxiccompatible substances employed in pharmaceutical compositions.

Pharmaceutical compositions are typically prepared by thoroughly andintimately mixing or blending the active agent with a pharmaceuticallyacceptable carrier and one or more optional ingredients. The resultinguniformly blended mixture may then be shaped or loaded into tablets,capsules, pills, canisters, cartridges, dispensers, and the like, usingconventional procedures and equipment.

In one embodiment, the pharmaceutical compositions are suitable for oraladministration. One exemplary dosing regimen would be an oral dosageform administered once or twice daily. Suitable compositions for oraladministration may be in the form of capsules, tablets, pills, lozenges,cachets, dragees, powders, granules; solutions or suspensions in anaqueous or non-aqueous liquid; oil-in-water or water-in-oil liquidemulsions; elixirs or syrups; and the like; each containing apredetermined amount of the active agent.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills, and the like), the composition will typicallycomprise the active agent and one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate. Solid dosageforms may also comprise: fillers or extenders, such as starches,microcrystalline cellulose, lactose, sucrose, glucose, mannitol, and/orsilicic acid; binders, such as carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, suchas glycerol; disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and/or sodium carbonate; solution retarding agents, such as paraffin;absorption accelerators, such as quaternary ammonium compounds; wettingagents, such as cetyl alcohol and/or glycerol monostearate; absorbents,such as kaolin and/or bentonite clay; lubricants, such as talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, and/or mixtures thereof; coloring agents; and buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants may also be presentin the pharmaceutical compositions. Exemplary coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate, polyvinyl acetatephthalate, hydroxypropyl methylcellulose phthalate, methacrylicacid-methacrylic acid ester copolymers, cellulose acetate trimellitate,carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetatesuccinate, and the like. Examples of pharmaceutically acceptableantioxidants include: water-soluble antioxidants, such as ascorbic acid,cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodiumsulfite, and the like; oil-soluble antioxidants, such as ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, lecithin,propyl gallate, alpha-tocopherol, and the like; and metal-chelatingagents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol,tartaric acid, phosphoric acid, and the like.

Compositions may also be formulated to provide slow or controlledrelease of the active agent using, by way of example, hydroxypropylmethyl cellulose in varying proportions or other polymer matrices,liposomes and/or microspheres. In addition, the pharmaceuticalcompositions of the invention may contain opacifying agents and may beformulated so that they release the active agent only, orpreferentially, in a certain portion of the gastrointestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes. The activeagent can also be in micro-encapsulated form, if appropriate, with oneor more of the above-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Liquid dosage formstypically comprise the active agent and an inert diluent, such as, forexample, water or other solvents, solubilizing agents and emulsifiers,such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Suspensions maycontain suspending agents such as, for example, ethoxylated isostearylalcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminium metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof the invention may be packaged in a unit dosage form. The term “unitdosage form” refers to a physically discrete unit suitable for dosing apatient, i.e., each unit containing a predetermined quantity of theactive agent calculated to produce the desired therapeutic effect eitheralone or in combination with one or more additional units. For example,such unit dosage forms may be capsules, tablets, pills, and the like.

In another embodiment, the compositions of the invention are suitablefor inhaled administration, and will typically be in the form of anaerosol or a powder. Such compositions are generally administered usingwell-known delivery devices, such as a nebulizer, dry powder, ormetered-dose inhaler. Nebulizer devices produce a stream of highvelocity air that causes the composition to spray as a mist that iscarried into a patient's respiratory tract. An exemplary nebulizerformulation comprises the active agent dissolved in a carrier to form asolution, or micronized and combined with a carrier to form a suspensionof micronized particles of respirable size. Dry powder inhalersadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. An exemplary dry powderformulation comprises the active agent dry-blended with an excipientsuch as lactose, starch, mannitol, dextrose, polylactic acid,polylactide-co-glycolide, and combinations thereof. Metered-doseinhalers discharge a measured amount of the active agent usingcompressed propellant gas. An exemplary metered-dose formulationcomprises a solution or suspension of the active agent in a liquefiedpropellant, such as a chlorofluorocarbon or hydrofluoroalkane. Optionalcomponents of such formulations include co-solvents, such as ethanol orpentane, and surfactants, such as sorbitan trioleate, oleic acid,lecithin, and glycerin. Such compositions are typically prepared byadding chilled or pressurized hydrofluoroalkane to a suitable containercontaining the active agent, ethanol (if present) and the surfactant (ifpresent). To prepare a suspension, the active agent is micronized andthen combined with the propellant. Alternatively, a suspensionformulation can be prepared by spray drying a coating of surfactant onmicronized particles of the active agent. The formulation is then loadedinto an aerosol canister, which forms a portion of the inhaler.

The crystalline compound of the invention can also be administeredparenterally (e.g., by subcutaneous, intravenous, intramuscular, orintraperitoneal injection). For such administration, the active agent isprovided in a sterile solution, suspension, or emulsion. Exemplarysolvents for preparing such formulations include water, saline, lowmolecular weight alcohols such as propylene glycol, polyethylene glycol,oils, gelatin, fatty acid esters such as ethyl oleate, and the like. Atypical parenteral formulation is a sterile pH 4-7 aqueous solution ofthe active agent. Parenteral formulations may also contain one or moresolubilizers, stabilizers, preservatives, wetting agents, emulsifiers,and dispersing agents.

These formulations may be rendered sterile by use of a sterileinjectable medium, a sterilizing agent, filtration, irradiation, orheat.

The crystalline compound of the invention can also be administeredtransdermally using known transdermal delivery systems and excipients.For example, the compound can be admixed with permeation enhancers, suchas propylene glycol, polyethylene glycol monolaurate,azacycloalkan-2-ones, and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

If desired, the crystalline compound of the invention may beadministered in combination with one or more other therapeutic agents.Thus, in one embodiment, compositions of the invention may optionallycontain other drugs that are co-administered with the crystallinecompound of the invention. For example, the composition may furthercomprise one or more drugs (also referred to as “secondary agents(s)”)selected from the group of anti-Alzheimer's agents, anticonvulsants(antiepileptics), antidepressants, anti-Parkinson's agents, dualserotonin-norepinephrine reuptake inhibitors (SNRIs), non-steroidalanti-inflammatory agents (NSAIDs), norepinephrine reuptake inhibitors,opioid agonists (opioid analgesics), opioid antagonists, selectiveserotonin reuptake inhibitors, sodium channel blockers, sympatholytics,and combinations thereof. Numerous examples of such therapeutic agentsare well known in the art, and examples are described herein. Bycombining the crystalline compound of the invention with a secondaryagent, triple therapy can be achieved, i.e., serotonin reuptakeinhibitory activity, norepinephrine reuptake inhibitory activity, andactivity associated with the secondary agent (e.g., antidepressantactivity), using only two active components. Since pharmaceuticalcompositions containing two active components are typically easier toformulate than compositions containing three active components, suchtwo-component compositions provide a significant advantage overcompositions containing three active components. Accordingly, in yetanother aspect of the invention, a pharmaceutical composition comprisesthe crystalline compound of the invention, a second active agent, and apharmaceutically acceptable carrier. Third, fourth, etc., active agentsmay also be included in the composition. In combination therapy, theamount of compound of the invention that is administered, as well as theamount of secondary agents, may be less than the amount typicallyadministered in monotherapy.

The crystalline compound of the invention may be either physically mixedwith the second active agent to form a composition containing bothagents; or each agent may be present in separate and distinctcompositions which are administered to the patient simultaneously orsequentially. For example, the crystalline compound of the invention canbe combined with a second active agent using conventional procedures andequipment to form a combination of active agents comprising thecrystalline compound of the invention and a second active agent.Additionally, the active agents may be combined with a pharmaceuticallyacceptable carrier to form a pharmaceutical composition comprising thecrystalline compound of the invention, a second active agent and apharmaceutically acceptable carrier. In this embodiment, the componentsof the composition are typically mixed or blended to create a physicalmixture. The physical mixture is then administered in a therapeuticallyeffective amount using any of the routes described herein.

Alternatively, the active agents may remain separate and distinct beforeadministration to the patient. In this embodiment, the agents are notphysically mixed together before administration but are administeredsimultaneously or at separate times as separate compositions. Suchcompositions can be packaged separately or may be packaged together in akit. When administered at separate times, the secondary agent willtypically be administered less than 24 hours after administration of thecompound of the invention, ranging anywhere from concurrent withadministration of the compound of the invention to about 24 hourspost-dose. This is also referred to as sequential administration. Thus,the crystalline compound of the invention can be orally administeredsimultaneously or sequentially with another active agent using twotablets, with one tablet for each active agent, where sequential maymean being administered immediately after administration of the compoundof the invention or at some predetermined time later (e.g., one hourlater or three hours later). Alternatively, the combination may beadministered by different routes of administration, i.e., one orally andthe other by inhalation.

In one embodiment, the kit comprises a first dosage form comprising thecrystalline compound of the invention and at least one additional dosageform comprising one or more of the secondary agents set forth herein, inquantities sufficient to carry out the methods of the invention. Thefirst dosage form and the second (or third, etc.) dosage form togethercomprise a therapeutically effective amount of active agents for thetreatment or prevention of a disease or medical condition in a patient.

Secondary agent(s), when included, are present in a therapeuticallyeffective amount, i.e., are typically administered in an amount thatproduces a therapeutically beneficial effect when co-administered withthe crystalline compound of the invention. The secondary agent can be inthe form of a pharmaceutically acceptable salt, solvate, optically purestereoisomer, and so forth. Thus, secondary agents listed below areintended to include all such forms, and are commercially available orcan be prepared using conventional procedures and reagents.

Representative anti-Alzheimer's agents include, but are not limited to:donepezil, galantamine, memantine, rivastigmine, selegiline, tacrine,and combinations thereof.

Representative anticonvulsants (antiepileptics) include, but are notlimited to: acetazolamide, albutoin, 4-amino-3-hydroxybutyric acid,beclamide, carbamazepine, cinromide, clomethiazole, clonazepam,diazepam, dimethadione, eterobarb, ethadione, ethosuximide, ethotoin,felbamate, fosphenytoin, gabapentin, lacosamide, lamotrigine, lorazepam,magnesium bromide, magnesium sulfate, mephenytoin, mephobarbital,methsuximide, midazolam, nitrazepam, oxazepam, oxcarbazepine,paramethadione, phenacemide, pheneturide, phenobarbital, phensuximide,phenytoin, potassium bromide, prcgabalin, primidone, progabide, sodiumbromide, sodium valproate, sulthiame, tiagabine, topiramate,trimethadione, valproic acid, valpromide, vigabatrin, zonisamide, andcombinations thereof. In a particular embodiment, the anticonvulsant isselected from carbamazepine, gabapentin, pregabalin, and combinationsthereof.

Representative antidepressants include, but are not limited to:adinazolam, amitriptyline, clomipramine, desipramine, dothiepin (e.g.,dothiepin hydrochloride), doxepin, imipramine, lofepramine, mirtazapine,nortriptyline, protriptyline, trimipramine, venlafaxine, zimelidine, andcombinations thereof.

Representative anti-Parkinson's agents include, but are not limited to:amantadine, apomorphine, benztropine, bromocriptine, carbidopa,diphenhydramine, entacapone, levodopa, pergolide, pramipexole,ropinirole, selegiline, tolcapone, trihexyphenidyl, and combinationsthereof.

Representative dual serotonin-norepinephrine reuptake inhibitors (SNRIs)include, but are not limited to: bicifadine, desvenlafaxine, duloxetine,milnacipran, nefazodone, venlafaxine, and combinations thereof.

Representative non-steroidal anti-inflammatory agents (NSAIDs) include,but are not limited to: acemetacin, acetaminophen, acetyl salicylicacid, alclofenac, alminoprofen, amfenac, amiprilose, amoxiprin,anirolac, apazone, azapropazone, benorilate, benoxaprofen, bezpiperylon,broperamole, bucloxic acid, carprofen, clidanac, diclofenac, diflunisal,diftalone, enolicam, etodolac, etoricoxib, fenbufen, fenclofenac,fenclozic acid, fenoprofen, fentiazac, feprazone, flufenamic acid,flufenisal, fluprofen, flurbiprofen, furofenac, ibufenac, ibuprofen,indomethacin, indoprofen, isoxepac, isoxicam, ketoprofen, ketorolac,lofemizole, lomoxicam, meclofenamate, meclofenamic acid, mefenamic acid,meloxicam, mesalamine, miroprofen, mofebutazone, nabumetone, naproxen,niflumic acid, nimesulide, nitroflurbiprofen, olsalazine, oxaprozin,oxpinac, oxyphenbutazone, phenylbutazone, piroxicam, pirprofen,pranoprofen, salsalate, sudoxicam, sulfasalazine, sulindac, suprofen,tenoxicam, tiopinac, tiaprofenic acid, tioxaprofen, tolfenamic acid,tolmetin, triflumidate, zidometacin, zomepirac, and combinationsthereof. In a particular embodiment, the NSAID is selected frometodolac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac,meloxicam, naproxen, oxaprozin, piroxicam, and combinations thereof. Ina particular embodiment, the NSAID is selected from ibuprofen,indomethacin, nabumetone, naproxen (for example, naproxen sodium), andcombinations thereof.

Representative muscle relaxants include, but are not limited to:carisoprodol, chlorzoxazone, cyclobenzaprine, diflunisal, metaxalone,methocarbamol, and combinations thereof.

Representative norepinephrine reuptake inhibitors include, but are notlimited to: atomoxetine, buproprion and the buproprion metabolitehydroxybuproprion, maprotiline, reboxetine (for example,(S,S)-reboxetine), viloxazine, and combinations thereof. In a particularembodiment, the norepinephrine reuptake inhibitor is selected fromatomoxetine, reboxetine, and combinations thereof.

Representative opioid agonists (opioid analgesics) and antagonistsinclude, but are not limited to: buprenorphine, butorphanol, codeine,dihydrocodeine, fentanyl, hydrocodone, hydromorphone, levallorphan,levorphanol, meperidine, methadone, morphine, nalbuphine, nalmefene,nalorphine, naloxone, naltrexone, nalorphine, oxycodone, oxymorphone,pentazocine, propoxyphene, tramadol, and combinations thereof. Incertain embodiments, the opioid agonist is selected from codeine,dihydrocodeine, hydrocodone, hydromorphone, morphine, oxycodone,oxymorphone, tramadol, and combinations thereof.

Representative selective serotonin reuptake inhibitors (SSRIs) include,but are not limited to: citalopram and the citalopram metabolitedesmethylcitalopram, dapoxetine, escitalopram (e.g., escitalopramoxalate), fluoxetine and the fluoxetine desmethyl metabolitenorfluoxetine, fluvoxamine (e.g., fluvoxamine maleate), paroxetine,sertraline and the sertraline metabolite demethylsertraline, andcombinations thereof. In certain embodiments, the SSRI is selected fromcitalopram, paroxetine, sertraline, and combinations thereof.

Representative sodium channel blockers include, but are not limited to:carbamazepine, fosphenytoin, lamotrignine, lidocaine, mexiletine,oxcarbazepine, phenytoin, and combinations thereof.

Representative sympatholytics include, but are not limited to: atenolol,clonidine, doxazosin, guanethidine, guanfacine, modafinil, phentolamine,prazosin, reserpine, tolazoline (e.g., tolazoline hydrochloride),tamsulosin, and combinations thereof.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

Exemplary Hard Gelatin Capsules for Oral Administration

The crystalline compound of the invention (50 g), spray-dried lactose(440 g) and magnesium stearate (10 g) are thoroughly blended. Theresulting composition is then loaded into hard gelatin capsules (500 mgof composition per capsule).

Alternately, the crystalline compound (20 mg) is thoroughly blended withstarch (89 mg), microcrystalline cellulose (89 mg) and magnesiumstearate (2 mg). The mixture is then passed through a No. 45 mesh U.S.sieve and loaded into a hard gelatin capsule (200 mg of composition percapsule).

Exemplary Gelatin Capsule Formulation for Oral Administration

The crystalline compound of the invention (100 mg) is thoroughly blendedwith polyoxyethylene sorbitan monooleate (50 mg) and starch powder (250mg). The mixture is then loaded into a gelatin capsule (400 mg ofcomposition per capsule).

Alternately, the crystalline compound (40 mg) is thoroughly blended withmicrocrystalline cellulose (Avicel PH 103; 259.2 mg) and magnesiumstearate (0.8 mg). The mixture is then loaded into a gelatin capsule(Size #1, White, Opaque) (300 mg of composition per capsule).

Exemplary Tablet Formulation for Oral Administration

The crystalline compound of the invention (10 mg), starch (45 mg) andmicrocrystalline cellulose (35 mg) are passed through a No. 20 mesh U.S.sieve and mixed thoroughly. The granules so produced are dried at 50-60°C. and passed through a No. 16 mesh U.S. sieve. A solution ofpolyvinylpyrrolidone (4 mg as a 10% solution in sterile water) is mixedwith sodium carboxymethyl starch (4.5 mg), magnesium stearate (0.5 mg),and talc (1 mg), and this mixture is then passed through a No. 16 meshU.S. sieve. The sodium carboxymethyl starch, magnesium stearate and talcare then added to the granules. After mixing, the mixture is compressedon a tablet machine to afford a tablet weighing 100 mg.

Alternately, the crystalline compound (250 mg) is thoroughly blendedwith microcrystalline cellulose (400 mg), silicon dioxide fumed (10 mg),and stearic acid (5 mg). The mixture is then compressed to form tablets(665 mg of composition per tablet).

Alternately, the crystalline compound (400 mg) is thoroughly blendedwith cornstarch (50 mg), croscarmellose sodium (25 mg), lactose (120mg), and magnesium stearate (5 mg). The mixture is then compressed toform a single-scored tablet (600 mg of compositions per tablet).

Exemplary Suspension Formulation for Oral Administration

The following ingredients are mixed to form a suspension containing 100mg of active agent per 10 mL of suspension:

Ingredients Amount Crystalline compound of the invention 1.0 g Fumaricacid 0.5 g Sodium chloride 2.0 g Methyl paraben 0.15 g Propyl paraben0.05 g Granulated sugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum ®K (magnesium aluminum silicate) 1.0 g Flavoring 0.035 mL Colorings 0.5mg Distilled water q.s. to 100 mL

Exemplary Injectable Formulation for Administration by Injection

The crystalline compound of the invention (0.2 g) is blended with 0.4 Msodium acetate buffer solution (2.0 mL). The pH of the resultingsolution is adjusted to pH 4 using 0.5 N aqueous hydrochloric acid or0.5 N aqueous sodium hydroxide, as necessary, and then sufficient waterfor injection is added to provide a total volume of 20 mL. The mixtureis then filtered through a sterile filter (0.22 micron) to provide asterile solution suitable for administration by injection.

Exemplary Compositions for Administration by Inhalation

The crystalline compound of the invention (0.2 mg) is micronized andthen blended with lactose (25 mg). This blended mixture is then loadedinto a gelatin inhalation cartridge. The contents of the cartridge areadministered using a dry powder inhaler, for example.

Alternately, a micronized compound of the invention (10 g) is dispersedin a solution prepared by dissolving lecithin (0.2 g) in demineralizedwater (200 mL). The resulting suspension is spray dried and thenmicronized to form a micronized composition comprising particles havinga mean diameter less than about 1.5 μm. The micronized composition isthen loaded into metered-dose inhaler cartridges containing pressurized1,1,1,2-tetrafluoroethane in an amount sufficient to provide about 10 μgto about 500 μg of the compound of the invention per dose whenadministered by the inhaler.

Alternately, the crystalline compound (25 mg) is dissolved in citratebuffered (pH 5) isotonic saline (125 mL). The mixture is stirred andsonicated until the compound is dissolved. The pH of the solution ischecked and adjusted, if necessary, to pH 5 by slowly adding aqueous 1Nsodium hydroxide. The solution is administered using a nebulizer devicethat provides about 10 μg to about 500 μg of the crystalline compoundper dose.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of the invention. These specific embodiments,however, are not intended to limit the scope of the invention in any wayunless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard meaning:

-   -   AcOH acetic acid    -   BSA bovine serum albumin    -   DCM dichloromethane (i.e., methylene chloride)    -   DIAD diisopropyl azodicarboxylate    -   DIPEA N,N-diisopropylethylamine    -   DMEM Dulbecco's Modified Eagle's Medium    -   DMSO dimethylsulfoxide    -   EDTA ethylenediaminetetraacetic acid    -   EtOAc ethyl acetate    -   EtOH ethanol    -   FBS fetal bovine serum    -   hDAT human dopamine transporter    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   hNET human norepinephrine transporter    -   hSERT human serotonin transporter    -   5-HT 5-hydroxytryptamine    -   IPA isopropyl alcohol    -   IPAc isopropyl acetate    -   MeCN acetonitrile (CH₃CN)    -   MeOH methanol    -   NA noradrenaline    -   PBS phosphate buffered saline    -   PPh₃ triphenylphosphine    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran    -   TsCl p-toluenesulfonyl chloride or 4-methylbenzenesulfonyl        chloride

Any other abbreviations used herein but not defined have their standard,generally accepted meaning. Unless noted otherwise, all materials, suchas reagents, starting materials and solvents, were purchased fromcommercial suppliers (such as Sigma-Aldrich, Fluka Riedel-de Haën, andthe like) and were used without further purification.

Preparation 14-(2-Methanesulfonyloxymethylphenyl)piperidine-1-carboxylic Acid t-ButylEster

4-(2-Carboxyphenyl)piperidine-1-carboxylic acid t-butyl ester (5.0 g,160 mmol, 1.0 eq.) and THF (100 mL, 1.0 mol) were combined at roomtemperature under nitrogen. 1.0M Borane-THF complex in THF (32.7 mL,32.7 mmol, 2.0 eq.) was added dropwise over 10 minutes (5° C. exotherm,gas evolution). The mixture was stirred at room temperature for 5minutes, then heated at 50° C. for 1 hour. The mixture was cooled toroom temperature, and the reaction was quenched slowly with MeOH (30 mL)(mild exotherm, significant gas evolution), then concentrated by rotaryevaporation. The material was azeotroped with MeOH (2×50 mL). The crudeproduct was dissolved in EtOAc (100 mL, 1 mol), washed with NaHCO₃ (50mL), then saturated aqueous NaCl (50 mL). The organic layer was driedover anhydrous Na₂SO₄, filtered, and concentrated in vacuo to yield4-(2-hydroxymethylphenyl)piperidine-1-carboxylic acid t-butyl ester (4.4g) as a clear, light yellow oil that solidified upon sitting.

4-(2-Hydroxymethylphenyl)piperidine-1-carboxylic acid t-butyl ester(50.0 g, 172 mmol, 1.0 eq.) was dissolved in DCM (500 mL, 8000 mmol).The mixture was cooled at 0° C. under nitrogen and methanesulfonicanhydride (44.8 g, 257 mmol, 1.5 eq.) was added in one portion. DIPEA(47.8 mL, 274 mmol, 1.6 eq.) was added dropwise over 5 minutes and themixture was stirred at 0° C. for 90 minutes. Water (400 mL, 20 mol) wasadded and the mixture was stirred for 5 minutes. The phases wereseparated, and the organic layer was washed with water (300 mL), driedover Na₂SO₄, and the solvent removed to yield the title compound (70 g)as a thick oil, which was used without further purification.

¹H NMR (400 MHz, DMSO-d₆) δ (ppm) 7.37-7.43 (m, 3H), 7.31 (d, 1H), 7.22(m, 2H), 5.38 (s, 2H), 4.28 (m, 2H), 2.92-3.10 (m, 1H), 2.92 (s, 3H),2.80-2.92 (m, 2H), 1.63-1.81 (m, 4H), 1.51 (s, 9H).

Example 1 Crystalline Hydrochloride Salt of4-[2-(2,4,6-Trifluorophenoxymethyl)phenyl]piperidine

4-(2-Methanesulfonyloxymethylphenyl)piperidine-1-carboxylic acid t-butylester (27.0 g, 60.6 mmol, 1.0 eq.) was dissolved in MeCN (540 mL) andadded to K₂CO₃ (25 g, 180 mmol, 3.0 eq.) and 2,4,6-trifluorophenol (13.5g, 90.9 mmol, 1.5 eq.). The mixture was vigorously stirred at 50° C. for6 hours, removed from the heat, and stirred overnight. The mixture wascooled at room temperature, and diluted with EtOAc (700 mL) and water(700 mL). The phases were separated, and the organic layer was washedtwice with 1.0 M NaOH in water (2×400 mL) and saturated aqueous NaCl(1×400 mL), then dried over Na₂SO₄ and the solvent removed to yieldcrude 4-[2-(2,4,6-trifluorophenoxymethyl)-phenyl]piperidine-1-carboxylicacid t-butyl ester (25.0 g). The crude product was combined with smallerscale runs for a total of 30 g, and purified by chromatography (0-10%EtOAc in hexanes) to yield4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine-1-carboxylic acidt-butyl ester (22.0 g).

The t-butyl ester (22.0 g, 31.3 mmol, 1.0 eq.) was combined with 1.25MHCl in EtOH (250 mL, 310 mmol, 10.0 eq.). The mixture was stirred atroom temperature for 8 hours, then stored at −10° C. over approximately48 hours. Most of solvent was removed by rotary evaporation. To theresulting thick slurry was added EtOAc (80 mL), followed by stirring atroom temperature for 2 hours. First crop was isolated by filtration, andthe filter cake was washed with EtOAc (20 mL) and dried to yield thetitle compound as a hydrochloride salt (8.5 g, >99% purity) white solid.HPLC of the filtrate shows ˜25% area of product. For the second crop,the solvent was removed by rotary evaporation and the resulting solid(˜10 g) was slurried in EtOAc (40 mL), first at room temperature, thenat 60° C., and again at room temperature to yield the title compound asa hydrochloride salt (1.7 g, >99% purity).

Two lots of the hydrochloride salt (18.5 g, 51.7 mmol) were combinedwith EtOAc (75 mL, 770 mmol). The resulting thick but free-flowingslurry was heated at 65° C. for 30 minutes, cooled to room temperature,and filtered. The flask and the filter cake were washed with EtOAc (20mL), and the solids dried under high vacuum at room temperatureovernight to yield the crystalline hydrochloride salt (18.2 g, 99.3%purity).

Good crystallinity was observed by XRPD. LC-MS (2 mg in 2 mL of 1:1MeCN:1M aq HCl; API 150EX LC/MS System) was found to be consistent withstructure. NMR (DMSO-d₆, Varian VnmrJ 400) was found to be consistentwith the structure and the salt form.

Example 2 Crystalline Hydrochloride Salt of4-[2-(2,4,6-Trifluorophenoxymethyl)phenyl]piperidine

Acetyl chloride (83.5 mL, 1170 mmol) was slowly added to EtOH (140 mL,2.4 mol).4-[2-(2,4,6-Trifluorophenoxymethyl)phenyl]piperidine-1-carboxylic acidt-butyl ester (55.0 g, 117 mmol) dissolved in EtOH (100 mL, 2.0 mol) wasadded and the resulting mixture was stirred at room temperature for 6hours. Most of solvent was removed by rotary evaporation. To theresulting thick slurry was added EtOAc (300 mL), followed by partialsolvent removal to ˜100 mL. Fresh EtOAc (200 mL) was added and theresulting slurry was stirred for 1 hour, filtered and dried to yield ahydrochloride salt (28.0 g, ˜99% purity). The filtrate was concentratedto a thick paste and IPAc (100 mL) was added, stirred for 1 hour,filtered and dried to further yield 5.0 g of the hydrochloride salt(˜99% purity).

Two lots of the hydrochloride salt (83.0 g, 230 mmol, ˜99% purity) werecombined with EtOAc (250 mL, 2.6 mol). The resulting slurry was heatedat 70° C. and then slowly cooled to room temperature, followed bystirring overnight. The resulting free-flowing slurry was filtered andthe filter cake was washed with EtOAc (50 mL) then dried under highvacuum for approximately 48 hours to yield a crystalline hydrochloridesalt (81.0 g, >99% purity). ¹H NMR (DMSO-d₆, 400 Hz) was found to beconsistent with the structure and the salt form of Example 1.

The crystalline hydrochloride salt (50.0 g, 1.40 mol, >99% purity) wasdissolved in IPA (250 mL, 3.3 mol), and the resulting slurry was heatedto 75° C. Water (25 mL, 1.4 mol) was added. Complete dissolution wasobserved in 5 minutes, and the internal temperature of the solution was65° C. The solution was slowly cooled to room temperature and thenstirred at room temperature overnight. The resulting solids werefiltered and dried under air for 2 hours to yield a semi-dry product.The solids were then dried under high vacuum at room temperature forapproximately 48 hours to yield the title crystalline hydrochloride salt(44.1 g, 99.5% purity). The material exhibited good crystallinity byXRPD and DSC.

Example 3 Crystalline Hydrochloride Salt of4-[2-(2,4,6-Trifluorophenoxymethyl)phenyl]piperidine

The title crystalline hydrochloride salt (151.1 g, 99.5% purity) wasalso prepared in a similar manner using 175.0 g of the hydrochloridesalt and 10 volumes of 5% water in IPA (total of 90 mL water and 1.8 LIPA).

Example 4 Powder X-Ray Diffraction

Powder X-ray diffraction patterns were obtained with a Rigaku MiniflexPXRD diffractometer using Cu Kα (30.0 kV, 15.0 mA) radiation. Theanalysis was performed with the goniometer running in continuous-scanmode of 2° (2θ) per min with a step size of 0.03° over a range of 2 to40° in two-theta angle. Samples were prepared on quartz specimen holdersas a thin layer of powdered material. The instrument was calibrated witha silicon metal standard, within ±0.02° two-theta angle. Arepresentative PXRD pattern for the crystalline hydrochloride salt ofExample 2 is shown in FIG. 1. The samples were hand ground prior totesting, in order to reduce particle size interferences to the relativeintensities.

The numerous intense powder diffraction peaks and relatively flatbaseline depicted in FIG. 1 strongly indicated that the crystallinehydrochloride salt of Example 2 possessed good crystallinity.

Powder X-ray diffraction patterns were also obtained for the crystallinehydrochloride salt of Example 1 and were found to be consistent withthose of the crystalline hydrochloride salt of Example 2.

Example 5 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-100 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Solutionssoftware. A 2.8 mg sample of the crystalline hydrochloride salt ofExample 2 was accurately weighed into a covered aluminum pan. After a 5minute isothermal equilibration period at 22° C., the sample was heatedusing a linear heating ramp of 10° C./min from 22° C. to 250° C. Arepresentative DSC thermograph is shown in FIG. 2.

The DSC thermograph demonstrates that the crystalline compound of theinvention has excellent thermal stability with a melting point at about196.9° C. and no thermal decomposition below 200.0° C.

A representative TGA trace is shown in FIG. 2, and indicates that asample of the crystalline form of Example 2 lost a small amount (about0.5%) of weight from room temperature to 150.0° C., which is consistentwith the loss of residual moisture or solvent.

A DSC thermograph and TGA trace were also obtained for the crystallinehydrochloride salt of Example 1 and were found to be consistent withthose of the crystalline hydrochloride salt of Example 2.

Example 6 Dynamic Moisture Sorption Assessment

A dynamic moisture sorption (DMS) assessment (also known as a moisturesorption-desorption profile) was performed for the crystallinehydrochloride salt of Example 2 using a VTI atmospheric microbalance,SGA-100 system (VTI Corp., Hialeah, Fla. 33016). A sample size ofapproximately 7.3 mg was used and the humidity was set at the ambientvalue at the start of the analysis. The DMS analysis consisted of a scanrate of 5% relative humidity/step over the full humidity range of 2%relative humidity to 90% relative humidity. The DMS run was performedisothermally at 25° C. A representative DMS profile is shown in FIG. 3.

The DMS profile demonstrates that the crystalline compound of theinvention has a reversible sorption/desorption profile withinsignificant hygroscopicity. The crystalline compound has a smallweight gain when it exposed to a broad humidity range from 2% relativehumidity up to 90% relative humidity, and less than about 2.0% weightgain when exposed to up to 90% relative humidity, which indicates thatthe crystalline form possesses only a minimal risk of hygroscopicity atambient conditions.

Example 7 Solid State Stability Assessment

A solid-state stability study was initiated with a representative lot ofthe crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine at −20° C., 5° C.,25° C./60% relative humidity (open container) and 40° C./75% relativehumidity (closed and open containers) storage conditions in glass. After28 days of storage, for the samples kept under all conditions, there wasno detectable change in the chemical purity, the chiral purity, PXRD,DSC, and TGA profiles.

Example 8 X-Ray Structure Analysis of a Single HCl Salt Crystal of4-[2-(2,4,6-Trifluorophenoxymethyl)phenyl]piperidine

A crystalline hydrochloride salt (105.0 mg, >99% purity) was preparedfrom EtOAc, as described in Example 2. The crystalline hydrochloridesalt was then combined with 5 mL of an IPA/water (10% water) solution.The mixture was shaken at room temperature (about 23° C.) until most ofthe material was dissolved in the solution. The solution was then placedon a heating plate which was pre-heated at 60° C. After 5 minutes, thesolution became clear indicating there was no any solid residue in theheated sample solution. The heating device was then turned off (totalheating time of about 10 minutes). The solution was allowed to stand forover two hours to allow the solution to slowly reach room temperature,at which point the solution was white and cloudy. The solution was thenchilled at 4° C. After about 7 days, large crystals were observed. Thecrystals were then isolated and dried.

The crystal structure was determined with a single crystal X-rayDiffractometer (Nonius Kappa-CCD Diffractometer equipped with OxfordCryostream Liquid Nitrogen Cooler using MoKα radiation). The size of thechunk crystal was 0.45×0.25×0.20 mm. A large crystal was cut along thecrystallographic dimension to obtain appropriate crystal size foranalysis (FIG. 5). Data was collected at a temperature of 294° K and120° K.

The crystal exhibited a monoclinic symmetry (P2₁/C space group), withthe following unit-cell parameters:

-   -   Axis (Å): a=11.631, b=7.057, c=42.532    -   Angle (°): B=104.595, A=C=90    -   V (Å³): 3378.4    -   Calculated density (g/cm³): 1.430

Reflection #: 20143

The crystals were found to consist of the4-[2-(2,4,6-trifluorophenoxymethyl)-phenyl]piperidine cation, chlorideanion and water molecules. In each unit cell, there were 2.56 watermolecules associated with eight drug molecule pairs (eight4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine cations and eightchloride anions), i.e., there was approximately one water molecule pereach three drug molecule pairs, where each drug molecule pair consistedof: one 4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine cation andone chloride anion. Thus, in one embodiment of the invention, about 0.32moles of water are present for every one mole of the4-[2-(2,4,6-trifluorophenoxymethyl)-phenyl]piperidine hydrochloridesalt; the formula for such a crystal can be written as: 14-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine. 1 HCl. 0.32 H₂O.

The crystal is built around the water molecule and the water content wasdetermined from the water occupancy factor in the unit cell. The wateroccupancy factor was calculated using the thermal motion parameters ofthe atoms and overall observed structure factors.

Assay 1 hSERT, hNET, and hDAT Binding Assays

Membrane radioligand binding assays were used to measure competitiveinhibition of labeled ligand (³H-citalopram or ³H-nisoxetine or³H-WIN35428) binding to membranes prepared from cells expressing therespective human recombinant transporter (hSERT or hNET or hDAT) inorder to determine the pK_(i) values of test compounds at thetransporters.

Membrane Preparation from Cells Expressing hSERT, hNET, or hDAT

Recombinant human embryonic kidney (HEK-293) derived cell lines stablytransfected with hSERT or hNET, respectively, were grown in DMEM mediumsupplemented with 10% dialyzed FBS (for hSERT) or FBS (for hNET), 100μg/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine and 250 μg/mlof the aminoglycoside antibiotic G418, in a 5% CO₂ humidified incubatorat 37° C. When cultures reached 80% confluence, the cells were washedthoroughly in PBS (without Ca²⁺ and Mg²⁺) and lifted with 5 mM EDTA inPBS. Cells were pelleted by centrifugation, resuspended in lysis buffer(10 mM Tris-HCl, pH7.5 containing 1 mM EDTA), homogenized, pelleted bycentrifugation, then resuspended in 50 mM Tris-HCl, pH 7.5 and 10%sucrose at 4° C. Protein concentration of the membrane suspension wasdetermined using a Bio-Rad Bradford Protein Assay kit. Membranes weresnap frozen and stored at −80° C. Chinese hamster ovary membranesexpressing hDAT (CHO-DAT) were purchased from PerkinElmer and stored at−80° C.

Binding Assays

Binding assays were performed in a 96-well assay plate in a total volumeof 200 μl assay buffer (50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4)with 0.5, 1, and 3 μg membrane protein, for SERT, NET and DAT,respectively. Saturation binding studies, to determine radioligand K_(d)values for ³H-citalopram, ³H-nisoxetine, or ³H-WIN35428, respectivelywere conducted using 12 different radioligand concentrations rangingfrom 0.005-10 nM (³H-citalopram); 0.01-20 nM (³H-nisoxetine) and 0.2-50nM (³H-WIN35428). Displacement assays for determination of pK_(i) valuesof test compounds were conducted with 1.0 nM ³H-citalopram, 1.0 nM³H-nisoxetine or 3.0 nM ³H-WIN35428, at 11 different concentrations oftest compound ranging from 10 pM to 100 μM.

Stock solutions (10 mM in DMSO) of test compound were prepared andserial dilutions made using Dilution Buffer (50 mM Tris-HCl, 120 mMNaCl, 5 mM KCl, pH 7.4, 0.1% BSA, 400 μM ascorbic acid). Non-specificradioligand binding was determined in the presence of 1 μM duloxetine, 1μM desipramine or 10 μM GBR12909 (each in Dilution Buffer) for thehSERT, hNET or hDAT assays, respectively.

Following a 60 minute incubation at 22° C. (or a period sufficient toreach equilibrium), the membranes were harvested by rapid filtrationover a 96-well UniFilter GF/B plate, pretreated with 0.3%polyethyleneimine, and washed 6 times with 300 wash buffer (50 mMTris-HCl, 0.9% NaCl, pH 7.5 at 4° C.). Plates were dried overnight atroom temperature, ˜45 μl of MicroScint™-20 (Perkin Elmer) added andbound radioactivity quantitated via liquid scintillation spectroscopy.Competitive inhibition curves and saturation isotherms were analyzedusing GraphPad Prism Software package (GraphPad Software, Inc., SanDiego, Calif.). IC₅₀ values were generated from concentration responsecurves using the Sigmoidal Dose Response (variable slope) algorithm inPrism GraphPad. K_(d) and B_(max) values for the radioligand weregenerated from saturation isotherms using the Saturation Binding GlobalFit algorithm in Prism GraphPad. pK_(i) (negative decadic logarithm ofK_(i)) values for test compounds were calculated from the best-fit IC₅₀values, and the K_(d) value of the radioligand, using the Cheng-Prusoffequation (Cheng & Prusoff (1973) Biochem. Pharmacol. 22(23):3099-3108):K_(i)=IC₅₀/(1+[L]/K_(d)), where [L]=concentration radioligand.

The compound of Example 1 (TFA salt) was tested in this assay and foundto exhibit a SERT pK_(i)≥7.9 and a NET pK_(i)≥8.0.

Assay 2 hSERT, hNET, and hDAT Neurotransmitter Uptake Assays

Neurotransmitter uptake assays were used to measure competitiveinhibition of ³H-serotonin (³H-5-HT), ³H-norepinephrine (³H-NE), and³H-dopamine (³H-DA) uptake into cells expressing the respectivetransporter (hSERT, hNET or hDAT) in order to determine the pIC₅₀ valuesof test compounds at the transporters.

³H-5-HT, ³H-NE, and ³H-DA Uptake Assays

HEK-293 derived cell lines stably-transfected with hSERT, hNET, or hDAT,respectively, were grown in DMEM medium supplemented with 10% dialyzedFBS (for hSERT) or FBS (for hNET and hDAT), 100 μg/ml penicillin, 100μg/ml streptomycin, 2 mM L-glutamine and 250 μg/ml of the aminoglycosideantibiotic G418 (for hSERT and hNET) or 800 ug/ml (for hDAT), in a 5%CO₂ humidified incubator at 37° C. When cultures reached 80% confluence,the cells were washed thoroughly in PBS (without Ca²⁺ and Mg²⁺) andlifted with 5 mM EDTA in PBS. Cells were harvested by centrifugation at1100 rpm for 5 minutes, washed once by resuspension in PBS, thencentrifuged. The supernatant was discarded and the cell pelletresuspended, by gentle trituration, in room temperature Krebs-Ringerbicarbonate buffer containing HEPES (10 mM), CaCl₂ (2.2 mM), ascorbicacid (200 μM) and pargyline (200 μM), pH 7.4. The final concentration ofcells in the cell suspension was 7.5×10⁴ cells/ml, 1.25×10⁵ cells/ml,and 5.0×10⁴ cells/ml for SERT, NET, and DAT cell lines, respectively.

Neurotransmitter uptake assays were performed in a 96-well assay platein a total volume of 400 μL assay buffer (Krebs-Ringer bicarbonatebuffer containing HEPES (10 mM), CaCl₂ (2.2 mM), ascorbic acid (200 μM)and pargyline (200 μM), pH 7.4) with 1.5×10⁴ and 2.5×10⁴ cells, for SERTand NET, respectively. Competition assays for determination of pIC₅₀values of test compounds were conducted with 11 differentconcentrations, ranging from 10 pM to 100 μM. Stock solutions (10 mM inDMSO) of test compound were prepared and serial dilutions prepared using50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4, 0.1% BSA, 400 μM ascorbicacid. Test compounds were incubated for 30 minutes at 37° C. with therespective cells, prior to addition of radiolabeled neurotransmitter,³H-5-HT (20 nM final concentration), ³H-NE (50 nM final concentration),or ³H-DA (100 nM final concentration). Non-specific neurotransmitteruptake was determined in the presence of 2.5 μM duloxetine or 2.5 μMdesipramine (each in Dilution Buffer) for the hSERT, hNET, or hDATassays, respectively.

Following a 10 minute incubation, at 37° C., with radioligand, the cellswere harvested by rapid filtration over a 96-well UniFilter GF/B plate,pretreated with 1% BSA, and washed 6 times with 650 μl wash buffer (icecold PBS). Plates were dried overnight at 37° C., ˜45 μl ofMicroScint™-20 (Perkin Elmer) added and incorporated radioactivityquantitated via liquid scintillation spectroscopy. Competitiveinhibition curves were analyzed using GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.). IC₅₀ values were generatedfrom concentration response curves using the Sigmoidal Dose Response(variable slope) algorithm in Prism GraphPad.

Assay 3 Ex Vivo SERT and NET Transporter Occupancy Studies

Ex vivo radioligand binding and neurotransmitter uptake assays were usedto determine the in vivo occupancy of SERT and NET, in selected brainregions, following in vivo administration (acute or chronic) of testcompounds. Following administration of test compound (by intravenous,intraperitoneal, oral, subcutaneous or other route) at the appropriatedose (0.0001 to 100 mg/kg), rats (≥n=4 per group) were euthanized atspecific time points (10 minutes to 48 hours) by decapitation and thebrain dissected on ice. Relevant brain regions were dissected, frozenand stored at −80° C. until use.

Ex Vivo SERT and NET Radioligand Binding Assays

For ex vivo radioligand binding assays, the initial rates of associationof SERT (³H-citalopram), and NET-(³H-nisoxetine) selective radioligandswith rat brain crude homogenates, prepared from vehicle and testcompound-treated animals, were monitored (see Hess et al. (2004) J.Pharmacol. Exp. Ther. 310(2):488-497). Crude brain tissue homogenateswere prepared by homogenizing frozen tissue pieces in 0.15 mL (per mgwet weight) of 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, pH 7.4 buffer.Radioligand association assays were performed in a 96-well assay platein a total volume of 200 μl assay buffer (50 mM Tris-HCl, 120 mM NaCl, 5mM KCl, 0.025% BSA, pH 7.4) with 650 μg wet weight tissue (equivalent to25 μg protein). Homogenates were incubated for up to 5 minutes with³H-citalopram (3 nM) and ³H-nisoxetine (5 nM), respectively, prior totermination of the assay by rapid filtration over a 96-well UniFilterGF/B plate, pretreated with 0.3% polyethyleneimine. Filters then werewashed 6 times with 300 μl wash buffer (50 mM Tris-HCl, 0.9% NaCl, pH7.4 at 4° C.). Non-specific radioligand binding was determined in thepresence of 1 μM duloxetine, or 1 μM despiramine, for ³H-citalopram or³H-nisoxetine, respectively. The plates were dried overnight at roomtemperature, ˜45 μl of MicroScint™-20 (Perkin Elmer) added and boundradioactivity quantitated via liquid scintillation spectroscopy. Theinitial rates of association of ³H-citalopram and ³H-nisoxetine weredetermined by linear regression using GraphPad Prism Software package(GraphPad Software, Inc., San Diego, Calif.). The average rate ofradioligand association to brain tissue homogenates from vehicle-treatedanimals was determined. The % occupancy of test compounds then wasdetermined using the following equation:% occupancy=100×(1−(initial rate association for test compound-treatedtissue/mean rate association for vehicle-treated tissue))ED₅₀ values were determined by plotting the log 10 of the dose of thetest compound against the % occupancy. ED₅₀ values were generated fromconcentration response curves using the Sigmoidal Dose Response(variable slope) algorithm in GraphPad Prism.

Ex Vivo SERT and NET Uptake Assays

Ex vivo neurotransmitter uptake assays, in which the uptake of ³H-5-HTor ³H-NE into rat brain crude homogenates, prepared from vehicle andtest compound-treated animals, were used to measure in vivo SERT and NETtransporter occupancy (see Wong et al. (1993) Neuropsychopharmacology8(1):23-33). Crude brain tissue homogenates were prepared byhomogenizing frozen tissue pieces in 0.5 mL (per mg wet weight) of 10 mMHEPES buffer pH 7.4, containing 0.32 M sucrose, 200 μM ascorbic acid and200 μM pargyline, at 22° C. Neurotransmitter uptake assays wereperformed in a 96-well Axygen plate in a total volume of 350 μl assaybuffer (Krebs-Ringer bicarbonate buffer with 10 mM HEPES, 2.2 mM CaCl₂,200 μM ascorbic acid and 200 μM pargyline, pH 7.4) with 50 μg protein.Homogenates were incubated for 5 minutes at 37° C. with ³H-5-HT (20 nM)and ³H-NE (50 nM), respectively, prior to termination of the assay byrapid filtration over a 96-well UniFilter GF/B plate, pretreated with 1%BSA. Plates were washed 6 times with 650 μl wash buffer (ice cold PBS)and dried overnight at 37° C., prior to addition of ˜45 μl ofMicroScint™-20 (Perkin Elmer) added. Incorporated radioactivity wasquantitated via liquid scintillation spectroscopy. Non-specificneurotransmitter uptake was determined in parallel assays in whichtissue homogenates were incubated with ³H-5-HT (20 nM) or ³H-NE (50 nM)for 5 minutes at 4° C.

Assay 4 Other Assays

Other assays that were used to evaluate the pharmacological propertiesof test compounds include, but are not limited to, cold ligand bindingkinetics assays (Motulsky and Mahan (1984) Molecular Pharmacol.25(1):1-9) with membranes prepared from cells expressing hSERT or hNET;conventional membrane radioligand binding assays using radiolabeled, forexample, tritiated, test compound; radioligand binding assays usingnative tissue from, for example rodent or human brain; neurotransmitteruptake assays using human or rodent platelets; neurotransmitter uptakeassays using crude, or pure, synaptosome preparations from rodent brain.

Assay 5 Formalin Paw Test

Compounds are assessed for their ability to inhibit the behavioralresponse evoked by a 50 μl injection of formalin (5%). A metal band isaffixed to the left hind paw of male Sprague-Dawley rats (200-250 g) andeach rat is conditioned to the band for 60 minutes within a plasticcylinder (15 cm diameter). Compounds are prepared in pharmaceuticallyacceptable vehicles and administered systemically (i.p., p.o.) atpre-designated times before formalin challenge. Spontaneous nociceptivebehaviors consisting of flinching of the injected (banded) hind paw arecounted continuously for 60 minutes using an automated nociceptionanalyzer (UCSD Anesthesiology Research, San Diego, Calif.).Antinociceptive properties of test articles are determined by comparingthe number of flinches in the vehicle and compound-treated rats (Yakshet al., “An automated flinch detecting system for use in the formalinnociceptive bioassay” (2001) J. Appl. Physiol. 90(6):2386-2402).

Assay 6 Spinal Nerve Ligation Model

Compounds are assessed for their ability to reverse tactile allodynia(increased sensitivity to an innocuous mechanical stimulus) induced bynerve injury. Male Sprague-Dawley rats are surgically prepared asdescribed in Kim and Chung “An experimental model for peripheralneuropathy produced by segmental spinal nerve ligation in the rat”(1992) Pain 50(3):355-363. Mechanical sensitivity is determined as the50% withdrawal response to innocuous mechanical stimuli (Chaplan et al.,“Quantitative assessment of tactile allodynia in the rat paw” (1994) J.Neurosci. Methods 53(1):55-63) before and after nerve injury. One tofour weeks post-surgery, compounds are prepared in pharmaceuticallyacceptable vehicles and administered systemically (i.p., p.o.). Thedegree of nerve injury-induced mechanical sensitivity before and aftertreatment serves as an index of the compounds' antinociceptiveproperties.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statues and regulations, all publications, patents andpatent applications cited herein are hereby incorporated by reference intheir entirety to the same extent as if each document had beenindividually incorporated by reference herein.

What is claimed is:
 1. A method of inhibiting norepinephrine reuptake ina mammal, the method comprising administering to the mammal anorepinephrine transporter-inhibiting amount of a crystallinehydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)-phenyl]piperidine characterized by(a) a powder x-ray diffraction pattern comprising diffraction peaks at2θ values of 4.44±0.20, 10.22±0.20, 17.16±0.20, and 21.78±0.20; or (b) adifferential scanning calorimetry trace which has a melting point ofabout 196.9° C.
 2. The method of claim 1, wherein the crystallinehydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized bya powder x-ray diffraction pattern comprising diffraction peaks at 2θvalues of 4.44±0.20, 10.22±0.20, 17.16±0.20, and 21.78±0.20.
 3. Themethod of claim 2, wherein the crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized byhaving one or more additional diffraction peaks at 2θ values selectedfrom 8.11±0.20, 13.18±0.20, 16.06±0.20, 18.38±0.20, 23.76±0.20,26.32±0.20, 27.24±0.20, 29.60±0.20, and 31.94±0.20.
 4. The method ofclaim 2, wherein the crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized bya powder x-ray diffraction pattern in which the peak positions aresubstantially in accordance with the peak positions of the pattern shownin FIG.
 1. 5. The method of claim 1, wherein the crystallinehydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized bya differential scanning calorimetry trace which has a melting point ofabout 196.9° C.
 6. The method of claim 5, wherein the crystallinehydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized bya differential scanning calorimetry trace substantially in accordancewith that shown in FIG.
 2. 7. The method of claim 1, wherein the mammalis a human.
 8. A method of inhibiting norepinephrine reuptake in amammal, the method comprising administering to the mammal a solidpharmaceutical composition comprising a pharmaceutically acceptablecarrier and a norepinephrine transporter-inhibiting amount of acrystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxy-methyl)phenyl]piperidine characterized by(a) a powder x-ray diffraction pattern comprising diffraction peaks at2θ values of 4.44±0.20, 10.22±0.20, 17.16±0.20, and 21.78±0.20; or (b) adifferential scanning calorimetry trace which has a melting point ofabout 196.9° C.
 9. The method of claim 8, wherein the crystallinehydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized bya powder x-ray diffraction pattern comprising diffraction peaks at 2θvalues of 4.44±0.20, 10.22±0.20, 17.16±0.20, and 21.78±0.20.
 10. Themethod of claim 9, wherein the crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized byhaving one or more additional diffraction peaks at 2θ values selectedfrom 8.11±0.20, 13.18±0.20, 16.06±0.20, 18.38±0.20, 23.76±0.20,26.32±0.20, 27.24±0.20, 29.60±0.20, and 31.94±0.20.
 11. The method ofclaim 9, wherein the crystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized bya powder x-ray diffraction pattern in which the peak positions aresubstantially in accordance with the peak positions of the pattern shownin FIG.
 1. 12. The method of claim 8, wherein the crystallinehydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized bya differential scanning calorimetry trace which has a melting point ofabout 196.9° C.
 13. The method of claim 12, wherein the crystallinehydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine is characterized bya differential scanning calorimetry trace substantially in accordancewith that shown in FIG.
 2. 14. The method of claim 8, wherein the mammalis a human.
 15. The method of claim 8, wherein the pharmaceuticalcomposition contains about 1 milligram to about 20 milligrams of thecrystalline hydrochloride salt of4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine.
 16. The method ofclaim 8, wherein the pharmaceutical composition contains about 1milligram to about 15 milligrams of the crystalline hydrochloride saltof 4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine.
 17. The methodof claim 8, wherein the pharmaceutical composition contains about 1milligram to about 10 milligrams of the crystalline hydrochloride saltof 4-[2-(2,4,6-trifluorophenoxymethyl)phenyl]piperidine.
 18. The methodof claim 8, wherein the pharmaceutical composition is administeredorally.